1. Field of Invention
This invention relates to molecules useful in determining minimal functional regions of IGFBP-3 and for antagonizing an IGF-I or IGF-II activity.
2. Description of Background and Related Art
The insulin-like growth factors I and II (IGF-I and IGF-II, respectively) mediate multiple effects in vivo, including cell proliferation, cell differentiation, inhibition of cell death, and insulin-like activity (reviewed in Clark and Robinson, Cytokine Growth Factor Rev., 7: 65-80 (1996); Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995)). Most of these mitogenic and metabolic responses are initiated by activation of the IGF-I receptor, an α2β2-heterotetramer closely related to the insulin receptor (McInnes and Sykes, Biopoly., 43: 339-366 (1998); Ullrich et al., EMBO J., 5: 2503-2512 (1986)). The IGF-I and insulin receptors bind their specific ligands with nanomolar affinity. IGF-I and insulin can cross-react with their respective non-cognate receptors, albeit at a 100-1000-fold lower affinity (Jones and Clemmons, supra). The crystal structure describing part of the extracellular portion of the IGF-I receptor has been reported (Garrett et al., Nature, 394: 395-399 (1998)).
Unlike insulin, the activity and half-life of IGF-I are modulated by six IGF-I binding proteins (IGFBPs 1-6), and perhaps additionally by a more distantly related class of proteins (Jones and Clemmons, supra; Baxter et al., Endocrinology, 139: 4036 (1998)). IGFBPs can either inhibit or potentiate IGF activity, depending on whether they are soluble or cell-membrane associated (Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995)). The IGFBPs bind IGF-I and IGF-II with varying affinities and specificities (Jones and Clemmons, supra; Bach and Rechler, supra). For example, IGFBP-3 binds IGF-I and IGF-II with a similar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-II with a much higher affinity than they bind IGF-I (Bach and Rechler, supra; Oh et al., Endocrinology, 132: 1337-1344 (1993)). WO 01/75064 discloses additional human secreted IGFBP-like polypeptides that are encoded by nucleic acid sequences isolated from cDNA libraries from adrenal gland mRNA and thymus mRNA.
Structurally, IGF-I is a single-chain, 70-amino-acid protein with high homology to proinsulin. Unlike the other members of the insulin superfamily, the C region of IGF's is not proteolytically removed after translation. The solution NMR structures of IGF-I (Cooke et al., Biochemistry, 30: 5484-5491 (1991); Hua et al., J. Mol. Biol., 259: 297-313 (1996)), mini-IGF-I (an engineered variant lacking the C-chain; DeWolf et al., Protein Science 5: 2193-2202 (1996)), and IGF-II (Terasawa et al., EMBO J., 13: 5590-5597 (1994); Torres et al., J. Mol. Biol., 248: 385-401 (1995)) have been reported. It is generally accepted that distinct epitopes on IGF-I are used to bind receptor and binding proteins. It has been demonstrated in animal models that receptor-inactive IGF mutants are able to displace endogenous IGF-I from binding proteins and thereby generate a net IGF-I effect in vivo (Loddick et al., Proc. Natl. Acad. Sci. USA, 95: 1894-1898 (1998); Lowman et al., Biochemistry, 37: 8870-8878 (1998); U.S. Pat. Nos. 6,121,416 and 6,251,865). While residues Y24, Y29, Y31, and Y60 are implicated in receptor binding, IGF mutants thereof still bind to IGFBPs (Bayne et al., J. Biol. Chem. 265: 15648-15652 (1990); Bayne et al., J. Biol. Chem., 264: 11004-11008 (1989); Cascieri et al., Biochemistry, 27: 3229-3233 (1988); Lowman et al., supra).
Additionally, a variant designated (1-27, gly4,38-70)-hIGF-I, wherein residues 28-37 of the C region of human IGF-I are replaced by a four-residue glycine bridge, has been discovered that binds to IGFPB's but not to IGF receptors (Bar et al., Endocrinology, 127: 3243-3245 (1990)).
A multitude of mutagenesis studies have addressed the characterization of the IGFBP-binding epitope on IGF-I (Bagley et al., Biochem. J., 259: 665-671 (1989); Baxter et al., J. Biol. Chem., 267: 60-65 (1992); Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988); Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990); Clemmons et al., Endocrinology, 131: 890-895 (1992); Oh et al., supra). In summary, the N-terminal residues 3 and 4 and the helical region comprising residues 8-17 were found to be important for binding to the IGFBPs. Additionally, an epitope involving residues 49-51 in binding to IGFBP-1, -2 and -5 has been identified (Clemmons et al., Endocrinology, supra, 1992). Furthermore, a naturally occurring truncated form of IGF-I lacking the first three N-terminal amino acids (called des (1-3)-IGF-I) was demonstrated to bind IGFBP-3 with 25 times lower affinity (Heding et al., J. Biol. Chem., 271: 13948-13952 (1996); U.S. Pat. Nos. 5,077,276; 5,164,370; and 5,470,828).
In an attempt to characterize the binding contributions of exposed amino acid residues in the N-terminal helix, several alanine mutants of IGF-I were constructed (Jansson et al., Biochemistry, 36: 4108-4117 (1997)). However, the circular dichroism spectra of these mutant proteins showed structural changes compared to wild-type IGF-I, making it difficult to clearly assign IGFBP-binding contributions to the mutated side chains. A different approach was taken in a very recent study where the IGFBP-1 binding epitope on IGF-I was probed by heteronuclear NMR spectroscopy (Jansson et al., J. Biol. Chem., 273: 24701-24707 (1998)). The authors additionally identified residues R36, R37 and R50 to be functionally involved in binding to IGFBP-1.
Other IGF-I variants have been disclosed. For example, in the patent literature, WO 96/33216 describes a truncated variant having residues 1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-I superagonists that are derivatives of the naturally occurring single-chain IGF-I having an abbreviated C domain. The IGF-I analogs are of the formula: BCn, A wherein B is the B domain of IGF-I or a functional analog thereof, C is the C domain of IGF-I or a functional analog thereof, n is the number of amino acids in the C domain and is from about 6 to about 12, and A is the A domain of IGF-I or a functional analog thereof.
Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988) discloses four mutants of IGF-I, three of which have reduced affinity to the Type 1 IGF receptor. These mutants are: (Phe23, Phe24, Tyr25) IGF-I (which is equipotent to human IGF-I in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu24) IGF-I and (Ser24) IGF-I (which have a lower affinity than IGF-I to the human placental Type 1 IGF receptor, the placental insulin receptor, and the Type 1 IGF receptor of rat and mouse cells), and desoctapeptide (Leu24) IGF-I (in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF-I, which has lower affinity than (Leu24)IGF-I for the Type 1 receptor and higher affinity for the insulin receptor). These four mutants have normal affinities for human serum binding proteins.
Bayne et al., J. Biol. Chem. 264: 11004-11008 (1988) discloses three structural analogs of IGF-I: (1-62) IGF-I, which lacks the carboxyl-terminal 8-amino-acid D region of IGF-I; (1-27,Gly4,38-70) IGF-I, in which residues 28-37 of the C region of IGF-I are replaced by a four-residue glycine bridge; and (1-27,Gly4,38-62) IGF-I, with a C region glycine replacement and a D region deletion. Peterkofsky et al., Endocrinology, 128: 1769-1779 (1991) discloses data using the Gly4 mutant of Bayne et al., supra, Vol. 264. U.S. Pat. No. 5,714,460 refers to using IGF-I or a compound that increases the active concentration of IGF-I to treat neural damage.
Cascieri et al., J. Biol. Chem. 264: 2199-2202 (1989) discloses three IGF-I analogs in which specific residues in the A region of IGF-I are replaced with the corresponding residues in the A chain of insulin. The analogs are: (Ile41,Glu45,Gln46,Thr49,Ser50,Ile51,Ser53,Tyr55,Gln56) IGF-I, an A chain mutant in which residue 41 is changed from threonine to isoleucine and residues 42-56 of the A region are replaced; (Thr49,Ser50,Ile51) IGF-I; and (Tyr55,Gln56)IGF-I.
Sliecker et al., Adv. Experimental Med. Biol., 343: 25-32 (1994)) describes the binding affinity of various IGF and insulin variants to IGFBPs, IGF receptor, and insulin receptor.
IGFBPs are secreted by cells in culture and either inhibit or enhance IGF-stimulated functions (Clemmons et al., (1991) In Modern Concepts of Insulin-like Growth Factors. E. M. Spencer, editor. Elsevier, New York, N.Y. 475-486). Known forms of IGFBPs include IGFBP-1, having a molecular weight of approximately 30-40 kDa in humans. See, e.g., WO89/09792, published Oct. 19, 1990, pertaining to cDNA sequences and cloning vectors for IGFBP-1 and IGFBP-2; WO89/08667, published Sep. 21, 1989, relating to an amino acid sequence of IGFBP-1; and WO89/09268, published Oct. 5, 1989, relating to a cDNA sequence of IGFBP-1 and methods of expression for IGFBP-1.
IGFBP-2 has a molecular weight of approximately 33-36 kDa. See, e.g., Binkert et al., The EMBO Journal, 8: 2497-2502 (1989), relating to a nucleotide and deduced amino acid sequence for IGFBP-2.
IGFBP-3 has a non-glycosylated molecular weight of about 28 kDa. See, e.g., Baxter et al., Biochim. Biophys. Res. Com., 139:1256-1261 (1986), pertaining to a glycosylated 53-kDa subunit of IGFBP-3 that was purified from human serum; Wood et al., Mol. Endocrinol., 2:1176-1185 (1988), relating to a full-length amino acid sequence for IGFBP-3 and cellular expression of the cloned IGFBP-3 cDNA in mammalian tissue culture cells; WO 90/00569, published Jan. 25, 1990, relating to isolating from human plasma an acid-labile subunit (ALS) of IGFBP complex and the particular amino acid sequence for ALS pertaining to a subunit of IGFBP-3; and Schmid et al., Biochim. Biophys. Res Com., 179: 579-585 (1991), relating to effects of full-length and truncated IGFBP-3 on two different osteoblastic cell lines.
Although initially some inconsistencies in nomenclature for IGFBP-4, IGFBP-5, and IGFBP-6 existed, in 1991 participants of the 2nd International IGF Symposium agreed upon an accepted IGFBP-4, IGFBP-5, and IGFBP-6 nomenclature. Using accepted terminology, Mohan et al., Proc. Natl. Acad. Sci., 86:8338-8342 (1989) relates to an N-terminal amino acid sequence for an IGFBP-4 isolated from medium conditioned by human osteosarcoma cells, and Shimasaki et al., Mol. Endocrinology, 4:1451-1458 (1990) pertains to IGFBP cDNAs encoding IGFBP-4 from rat and human. WO92/03471 published Mar. 5, 1992, relates to an IGFBP-4 (originally designated therein as IGFBP-5); and WO92/03470 published Mar. 5, 1992 relates to genetic material encoding IGFBP-4 (originally designated therein as IGFBP-5).
WO92/12243 published Jul. 23, 1992, relates to IGFBP-5 (originally designated therein as IGFBP-6). Andress and Birnbaum, Biochim. Biophys. Res Com., 176: 213-218 (1991) relates to the modulation of cellular action of a mixture of affinity-purified IGFBPs from U-2-cell-conditioned media on IGFs. WO92/03469 published Mar. 5, 1992, relates to genetic material encoding IGFBP-6 (originally designated therein as IGFBP-4); and WO92/03152 published Mar. 5, 1992, relates to an IGFBP-6 (originally designated therein as IGFBP-4). See also US Appln. No. 2003/0082744 as well as U.S. Pat. Nos. 6,025,465 and 5,212,074, which disclose IGFBP-6 and its fragments.
Zapf et al., J. Biol. Chem., 265:14892-14898 (1990) pertains to four IGFBPs (IGFBP-2, IGFBP-3, a truncated form of IGFBP-3, and IGFBP-4) isolated from adult human serum by insulin-like growth factor (IGF) affinity chromatography and high-performance liquid chromatography. Shimasaki et al., 2nd International IGF Symposium Abstract (January 1991) discusses amino-terminal amino acids for IGFBP-4, IGFBP-5, and IGFBP-6.
When administered alone, i.e., without any IGF, the IGFBPs may also be therapeutically useful for blocking the adverse effects of IGFs, such as those that occur when IGFs are produced in excess, e.g. free IGFs secreted by certain cancer cells such as hormone-producing cancer cells such as breast or kidney cancer cells. More recently, it was demonstrated that U-2 human osteosarcoma cells secrete IGFBP-5 and IGFBP-6 (Andress and Birnbaum, supra; Shimasaki et al., J. Biol. Chem. 266: 10646-10653 (1991); Shimasaki et al., Mol. Endocrinol., 5: 938-948 (1991)). Although affinity-purified IGF-binding proteins derived from U-2-conditioned medium clearly enhanced IGF-I stimulated mitogenesis (Andress and Birnbaum, supra), it was unclear from those studies which protein was responsible for this effect. Mohan et al. demonstrated that IGFBP-4, purified from TE-89 human osteosarcoma cells, inhibits IGF-stimulated osteoblast mitogenesis (Mohan et al., Proc. Natl. Acad. Sci. (U.S.A.), 86: 8338-8342 (1989); see also LaTour et al., Mol. Endocrinol., 4: 1806-1814 (1990)). WO 03/068160 discloses use of IGFBP-3 for inhibiting tumor growth. WO 03/006029 discloses a method of inducing apoptosis in a cancer cell comprising increasing the expression of IGFBP-5 by the cell to an apoptosis-inducing amount. A method of killing cancer cells, a method of sensitizing cancer cells to agents that induce apoptosis, and a method of treating cancer in a patient are also described. U.S. Pat. No. 6,410,335 discloses a method of predicting risk for prostate cancer, and US Application 2001/0018190 published Aug. 30, 2001 discloses a method of treating prostate cancer with, inter alia, IGFBPs, including IGFBP-3. U.S. Pat. No. 5,840,673 and EP 871,475 disclose a method of inhibiting growth of p53-related tumors by administering IGFBP-3 or a modulator of IGFBP-3 that upregulates IGFBP-3 expression or activity. WO 00/35473 discloses use of IGFBP-6 to treat inflammatory diseases including tumor angiogenesis. WO 94/22466 discloses use of any IGFBP-3 to treat cancer, including prostate cancer. WO 92/14834 discloses two IGFBPs isolated from rat serum, one identified as IGFBP-5 useful in treating cancer.
Exploitation of the interaction between IGF and IGFBP in screening, preventing, or treating disease has been limited, however, because of a lack of specific antagonists. The application of an IGF-1/IGF-2 antagonist as a potential therapeutic adjunct in the treatment of cancer is described by Pietrzkowski et al., Cancer Res., 52: 6447-6451 (1992). In that report, a peptide corresponding to the D-region of IGF-1 was synthesized for use as an IGF-1/2 antagonist. This peptide exhibited questionable inhibitory activity against IGF-1. The basis for the observed inhibition is unclear, as the D-region does not play a significant role in IGF-1 receptor (IGF-1R) binding but rather, in IGF-1 binding to the insulin receptor (Cooke et al., Biochem., 30: 5484-5491 (1991); Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988); Yee et al., Cell Growth and Different. 5: 73-77 (1994)). IGF antagonists whose mechanism of action is via blockade of interactions at the IGF-1 receptor interface may also significantly alter insulin action at the insulin receptor, a disadvantage of such antagonists.
Certain IGF-1 antagonists have also been described by WO 00/23469, which discloses the portions of IGFBP and IGF peptides that account for IGF-1 and IGFBP binding, i.e., an isolated IGF binding domain of an IGFBP or modification thereof that binds IGF with at least about the same binding affinity as the full-length IGFBP. The patent publication also discloses an IGF antagonist that reduces binding of IGF to an IGF receptor, and/or binds to a binding domain of IGFBP.
Additionally, EP 639981 discloses pharmaceutical compositions comprising short peptides that function as IGF-1 receptor antagonists. The peptides used in the pharmaceutical compositions consist of less than 25 amino acids, comprise at least a portion of the C or D region from IGF-1, and inhibit IGF-1-induced autophosphorylation of IGF-1 receptors. Methods of inhibiting cell proliferation and of treating individuals suspected of suffering from or susceptible to diseases associated with undesirable cell proliferation such as cancer, restenosis, and asthma are disclosed.
Generation of specific IGF-1 antagonists has been restricted, at least in part, because of difficulties in studying the structure of IGF and IGFBP. Due to the inability to obtain crystals of IGF-1 suitable for diffraction studies, for example, an extrapolation of IGF-1 structure based on the crystal structure of porcine insulin was the most important structural road map for IGF-1 available (Blundell et al., Proc. Natl. Acad. Sci. USA, 75:180-184 (1978)). See also Blundell et al., Fed Proc., 42: 2592 (1983), which discloses tertiary structures, receptor binding, and antigenicity of IGFs. Based on studies of chemically modified and mutated IGF-1, a number of common residues between IGF-1 and insulin have been identified as being part of the IGF-1R-insulin receptor contact site, in particular the aromatic residues at positions 23-25. Using NMR and restrained molecular dynamics, the solution structure of IGF-1 was recently reported (Cooke et al., supra). The resulting minimized structure was shown to better fit the experimental findings on modified IGF-1, as well as the extrapolations made from the structure-activity studies of insulin. Further, De Wolf et al., Protein Sci., 5: 2193 (1996) discloses the solution structure of a mini-IGF-1. Sato et al., Int. J. Pept., 41: 433 (1993) discloses the three-dimensional structure of IGF-1 determined by 1 H-NMR and distance geometry. Torres et al., J. Mol. Biol. 248: 385 (1995) discloses the solution structure of human IGF-2 and its relationship to receptor and binding protein interactions. Laajoki et al., J. Biol. Chem. 275: 10009 (2000) discloses the solution structure and backbone dynamics of long-(Arg(3)) IGF-1.
Peptide sequences capable of binding to insulin and/or insulin-like growth factor receptors with either agonist or antagonist activity and identified from various peptide libraries are described in WO 01/72771 published Oct. 4, 2001.
US Application No. 2003/0092631 published May 15, 2003 discloses peptides that antagonize the interaction of IGF-1 with its binding proteins, insulin receptor, and IGF receptor. These IGF antagonist peptides are useful in treating disorders involving IGF-1 as a causative agent, such as, for example, various cancers.
Regarding the structural information on the classical IGFBPs, they have a molecular mass ranging from 22 to 31 kDa and contain a total of 16-20 cysteines in their conserved amino- and carboxy-terminal domains (Bach and Rechler, supra; Clemmons, Cytokine Growth Factor Rev., 8: 45-62 (1997); Martin and Baxter, Curr. Op. Endocrinol. Diab., 16-21 (1994)). The central domain connecting both cysteine-rich regions is only weakly conserved and contains the cleavage sites for IGFBP-specific proteases (Chernausek et al., J. Biol. Chem., 270: 11377-11382 (1995); Clemmons, supra; Conover, Prog. Growth Factor Res., 6: 301-309 (1995)). Further regulation of the IGFBPs may be achieved by phosphorylation and glycosylation (Bach and Rechler supra; Clemmons, supra). There is no high-resolution structure available for any intact member of the IGFBP family. U.S. Pat. No. 6,500,635 discloses IGFBP-5 and its variants. U.S. Pat. Nos. 6,391,588 and 6,489,294 disclose truncated C-terminal IGFBP-5 fragments with reduced affinity for IGF-I as compared to full-length IGFBP-5. U.S. Pat. No. 6,369,029 discloses stimulating osteogenesis with C-terminal-truncated IGFBP-5. These compounds may be used for stimulating bone cell growth, for treating a bone disorder, or for stimulating mitogenic activity.
The NMR structures of two N-terminal fragments from IGFBP-5 that retain IGF-binding activity have recently been reported, showing that residues 40-92 of IGFBP-5 comprise the IGF binding site in the N-terminal domain of that protein (Kalus et al., EMBO J., 17: 6558-6572 (1998)). Other studies have found that N-terminal fragments (residues 1-88 and 1-97) of IGFBP-3 are also able to bind IGFs (Galanis et al., Journal of Endocrinology, 169 (1): 123-133 (2001); Vorwerk et al., Endocrinology, 143 (5): 1677-1685 (2002)).
In particular, Galanis et al. have synthesized both the amino-terminal (residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165) domains of human IGFBP-3 in bacteria, as fusion proteins with a carboxyl-terminal FLAG peptide. Although only C-165 showed binding to IGF-I and IGF-II by solution-binding assays, both N-88 and C-165 demonstrated binding to IGF-I and -II by biosensor analysis albeit with reduced affinities compared with full-length IGFBP-3. Only the carboxyl-terminal fragment (C-165) was able to form hetero-trimeric complexes with IGF-I and the acid-labile subunit (ALS).
Vorwerk et al. measured the binding of IGF-I and IGF-II to recombinant human N-terminal (residues 1-97; N-97) and C-terminal (residues 98-264; C-98) IGFBP-3 fragments and compared it with IGF binding to intact IGFBP-3 using biosensor analysis. Experiments were carried out either with binding protein or fragment immobilized or with IGF immobilized. These experiments showed that IGF-I and IGF-II bind to IGFBP-3 with affinities of 4-5×10−9 M and similar binding kinetics. The affinities of both N-97 and C-98 for IGF proteins were approximately three orders of magnitude less than that of full-length IGFBP-3.
US Application No. 2003/0161829A1 published Aug. 28, 2003 and WO 03/025121 disclose fragments of IGFBP-3 that do not bind IGF-I to treat conditions characterized by immune stimulation rather than deficiency. The peptides target the CD74-homology domain sequence at the C-terminus of IGFBP-3 and activities localized to that region, having unique antigenicity. Peptides made to sequences in this region have previously been shown to interfere with the binding of IGFBP-3 to a number of its known ligands, including RXR-alpha, transferrin, ALS, plasminogen, fibrinogen and pre-kallikrein (Liu et al., J. Biol. Chem., 275: 33607-33613 (2000); Weinzimer et al., J. Clin. Endocrinol. Metab., 86: 1806-13 (2001); Campbell et al., Am. J. Physiol., 275: E321-E231 (1998); Campbell et al., J. Biol. Chem., 274: 30215-30221 (1999); Firth, et al., J. Biol. Chem., 273: 2631-2638, (1998)).
The IGFBP-3-derived metal-binding domain peptides differ from previously disclosed IGFBP-3-derived molecules including in their inability to bind IGF-I, their unique antigenicity, and the absence of the IGFBP-3 putative death receptor (P4.33) interaction domain of IGFBP-3 (so-called “mid-region”; amino acids 88-148). The P4.33 putative death receptor is described in WO 01/87238 (Genbank Accession Number BC031217; gi:21411477). For example, WO 02/34916 teaches the use of point mutants of IGFBP-3 in which the binding to IGF-I is impaired. However, the described molecules contain the mid-region of IGFBP-3 and would be expected to exert biological effects by interacting with the P4.33 putative receptor. WO 01/87238 teaches the use of P4.33 modulators for treating disease. The metal-binding peptides do not include the P4.33 putative interaction domain (mid-region of IGFBP-3).
U.S. Pat. No. 6,417,330, WO 99/63086, and US application no. 2002/0072589 disclose IGFBP-3 variants modified to be resistant to hydrolysis. Also disclosed are variant IGFBP-3s where the nuclear localization signal (NLS) in native IGFBP-3 is altered. Additionally, amino-terminally extended IGFBP-3s are disclosed that include a variety of N-terminal extensions, including peptide and nucleotide binding domains, specific binding members such as ligand-binding domains from receptors or antigen-binding domains from immunoglobins, and peptide and protein hormones and growth factors. N-terminally extended IGFBP-3s may comprise hydrolysis-resistant or NLS-variant IGFBP-3s.
Some recent publications have described the use of IGFBP-3 peptides to treat cells in culture. The only peptides found to be active on breast cancer cells are derived from the mid-region of IGFBP-3 (McCaig et al., Br. J. Cancer, 86: 1963-1969 (2002); Perks et al. Biochim. Biophys. Res. Comm., 294: 988-994 (2002)).
US Application No. 2003/0059430 published Mar. 27, 2003 discloses that the IGF-binding protein-derived peptides described above, including short peptides containing just 12-22 amino acids from the C-terminal domain of IGFBP-3, can mimic the full molecule's co-apoptotic, cell-penetrating, and metal-binding properties.
WO 03/052079 discloses mutants of IGFBP-3 that can inhibit DNA synthesis, can induce apoptosis, bind to neither human IGF-I nor human IGF-II, and comprise a mutation at Y57.
WO 02/098914 discloses a crystal suitable for X-ray diffraction, comprising a complex of IGF-I or -II and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9th to 12th cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7th to 10th cysteine of IGFBP-6; methods for the determination of the atomic coordinates of such a crystal; IGFBP mutants with enhanced binding affinity for IGF-I and/or IGF-II, and methods to identify and optimize small molecules that displace IGFs from their binding proteins.
WO 02/34916 discloses mutant IGFBP-3 polypeptides and fragments thereof that have either no binding, or diminished binding to IGFs, yet retain their ability to bind to the human IGFBP-3 receptor (“P4.33”). The fragments are N-deletion fragments with 87 to 264 amino acids. The fragment 1-87 binds poorly to IGF-I, and the other fragments (1-46, 1-75, and 1-80) do not bind at all.
WO 00/23469 discloses IGFBP fragments that account for IGF-IGFBP binding. It provides an isolated IGF binding domain of an IGFBP or modifications thereof, which binds IGF with at least about the same binding affinity as the full-length IGFBP. It also provides an IGF antagonist that reduces binding of IGF to an IGF receptor. It especially relates to IGFBP-2 fragments, but also provides the isolated IGF binding domains of IGFBP-1, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6. As with the IGF binding domain of IGFBP-2, the amino acid sequences comprising the IGF binding domain of the other IGFBPs could include modified forms so long as the binding affinity of the binding domain is about the same as that of the comparable native full-length IGFBP.
WO 99/32620 discloses IGFBP fragments and utilization thereof, i.e., peptides that are characterized in that the amino acid sequence parts thereof correspond to the amino acid sequence of IGFBP. The invention also relates to cyclic, glycosylated, phosphorylated, acetylated, amidated and/or sulfatized derivatives. These include C-terminal domains of IGFBP-3.
The use of gene fusions, though not essential, can facilitate the expression of heterologous peptides in E. coli as well as the subsequent purification of those gene products (Harris, in Genetic Engineering, Williamson, R., Ed. (Academic Press, London, Vol. 4, 1983), p. 127; Ljungquist et al., Eur. J. Biochem., 186: 557-561 (1989); and Ljungquist et al., Eur. J. Biochem., 186: 563-569 (1989)). Protein A fusions are often used because the binding of protein A, or more specifically the Z domain of protein A, to IgG provides an “affinity handle” for the purification of the fused protein. It has also been shown that many heterologous proteins are degraded when expressed directly in E. coli, but are stable when expressed as fusion proteins (Marston, Biochem J., 240: 1 (1986)).
Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly residue. Using standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5′ end of the gene encoding the desired peptide.
Alternatively, one can employ proteolytic cleavage of fusion proteins (Carter, in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch et al., eds. (American Chemical Society Symposium Series No. 427, 1990), Ch 13, pages 181-193)).
There is a continuing need in the art for a molecule that can be used to elucidate binding epitopes on IGFBP-3 and other ligands, acts as an IGF antagonist to control the levels of circulating IGF as well as receptor response, for therapeutic or diagnostic uses, and can be used for other therapeutic, diagnostic, or assay purposes.